Create a custom analysis workflow for use with Ion AmpliSeq™ HD panels

To analyze Ion AmpliSeq™ HD sequencing data in Ion Reporter™ Software, you must create a custom analysis workflow for use with your Ion AmpliSeq™ HD panels. To create a custom analysis workflow, you must first copy one of the preinstalled Ion AmpliSeq™ HD analysis workflow templates, then add target regions files, and any available hotspots files or CNV baseline. For RNA samples, you must also add fusion panel files to your custom analysis workflow.

After you create the custom analysis workflow, you can select the analysis workflow in Torrent Suite™ Software to transfer data automatically to the appropriate Ion Reporter™ Server and use the analysis workflow for the data analysis in Ion Reporter™ Software.

  1. In the Workflows tab, click Overview.

    For information on available Ion AmpliSeq™ HD analysis workflow templates, see Analysis workflow templates for Ion AmpliSeq HD.

  2. In the Workflows table, select the Ion AmpliSeq™ HD analysis workflow template that you want to copy, then click  Actions > Copy.

    The Edit workflow bar opens to the Research Application step with the Research Application and Sample Group preselected.

    Note: When you copy analysis workflow templates, you cannot change these settings.

  3. Click Next, then in the Reference step, select or upload the appropriate files.

    Option

    Description

    Select a file that has been previously uploaded to Ion Reporter™ Software.

    Select the Target Regions and, optionally, Hotspot Regions files from the dropdown lists.

    Upload the entire package of the Ion AmpliSeq™ HD panel files directly from AmpliSeq.com.

    Note: When you use this option, a target regions file and any available hotspot regions or fusion files specific for your panel are uploaded.

    Click AmpliSeq Import.

    Import a target regions file that you previously downloaded from AmpliSeq.com and saved to your local storage.

    1. Under the Target Regions list, click Upload.

    2. Click Select file, browse to, then select the target regions BED file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

    (Optional) Upload a hotspot regions file that was previously uploaded from AmpliSeq.com and saved to your local storage.

    1. Click Upload under the Hotspots Regions list.

    2. Click Select file, browse to, then select the hotspot regions BED file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

    If you use a panel that detects fusions, upload a FASTA file that was previously uploaded from AmpliSeq.com and saved to your local storage.

    1. Click Upload under the Fusion Panel list.

    2. Click Select file, browse to, then select the fusions ZIP file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

  4. Click Next.
  5. In the Annotation step, confirm or select an annotation set, confirm or select a MyVariants database, then click Next.
  6. In the Filters step, select a filter chain from the Filter Chains list to change the default filter chain for analyses that use this analysis workflow. If you do not change the filter chain, the default filter chain is saved for the new analysis workflow.

    Option

    Description

    Called Variants and Controls

    This is the default filter chain for Ion AmpliSeq™ HD analysis workflows. Use this filter chain for analysis results that report all variants (either hotspots or novel) that pass the filter and are not called as reference or NOCALL.

    Variant types include: SNV, INDEL, MNV, CNV, LONGDEL, FUSION, EXPR_CONTROL_ASSAYS_5P_3P, RNA_HOTSPOT, GENE_EXPRESSION, RNAExonVariant, ProcControl, and FLT3ITD.

    Called Hotspot Variants and Controls

    Select this filter chain for analysis results that report all hotspot variants that pass the filter and are not called as reference or NOCALL.

    Filter variant types include: SNP and INDEL.

    Variant Matrix Summary

    Select this filter chain for analysis results that replicate data that is shown for Ion AmpliSeq™ HD analysis results in the Variant Matrix Summary.

    Variant types include: SNV/INDEL, CNV, fusions, and RNAExonVariants.

    Oncomine™ Variants (5.10 or later)

    Select this filter chain to show only the variants that are annotated with the Oncomine™ Variant Annotator plugin. For more information, see Oncomine Variant Annotator plugin criteria.

  7. Click Next.
  8. IMPORTANT! To add a copy number baseline, contact your Field Bioinformatics Specialist (FBS).

    In the Copy Number step, select a copy number baseline from the Baseline list, if applicable, then click Next.
  9. In the Plugins step select a plugin, then click Next.

    In the Plugins step, the Oncomine™ Variant Annotator plugin is automatically selected. This plugin adds annotations for variants that are relevant to cancer with Oncomine™ Gene Class and Oncomine™ Variant Class information. This plugin is included by default with the Ion AmpliSeq™ HD analysis workflow templates. If you import the VCF file of analysis results into Oncomine™ Reporter, these annotations are included in a report that is generated from that software.

  10. In the Final Report step, select or confirm the final report template that is selected in the list, then click Next.
  11. In the Parameters step, make any required changes, then click Next.

    IMPORTANT! If you are using the Ion AmpliSeq™ HD test panel, consult your field support representative and other training materials before you change the parameters. If you designed a white glove panel, consult your white glove representative or field support representative to determine if parameter changes are required.

  12. In the Confirm step, name the analysis workflow, enter an optional description, then click Save Workflow.

The newly created analysis workflow is added to the list of analysis workflows in the Workflows tab in the Overview screen.